Maternal exposure to a high fat (HF) diet during pregnancy increases estrogen receptor (ER+) and ER- mammary cancer risk among female offspring in animal models and in humans. The effect may not be limited to F1 generation daughters: we found that an exposure during pregnancy to a diet containing ethinyl estradiol (EE2) increased mammary cancer risk also in granddaughters (F2 generation) and great granddaughters (F3 generation). Since HF diet increases pregnancy E2 levels, we are proposing to investigate in mice whether maternal exposure to HF diet increases the risk of developing ER+ and/or ER- mammary cancer in F1-F4 generation offspring. In addition, we will investigate whether these transgenerational effects involve changes in DNA methylation. Our preliminary analysis performed using massively parallel sequencing identified 144 named genes which were either hypo- or hypermethylated in the mammary glands of F1-F3 generation offspring of EE2 exposed dams, compared to controls. 21% of these genes were polycomb target genes (PcGTs), which in turn included some tumor suppressor genes (TSGs), suggesting that maternal diet during pregnancy, including consumption of a HF diet, may induce methylation of PcTGs/TSGs in the offspring's breast. Interestingly, women at high risk of developing breast cancer exhibit methylation of PcGTs and TSGs. The increase in DNA methylation may be caused by up-regulation of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and polycombs (EZH2, SUZ12), which we and others have found to occur in the offspring of estrogen exposed dams. Further, up-regulation of DNMTs and polycombs may have been initiated by estrogen-induced suppression of non-coding miRNAs which target them, as seen in MCF-7 human breast cancer cells and mammary glands of rats exposed to EE2 or HF diet in utero (our preliminary data). In this study, we test a hypothesis that maternal exposure to a HF diet during pregnancy induces a transgenerational increase in mammary cancer risk in the F1-F4 generation offspring by inducing DNA methylation of PcTGs/TSGs, via estrogen-induced down-regulation of miRNAs. A causal chain involving estrogen-induced down-regulation of miRNA, up-regulation of DNMTs and polycombs and subsequent methylation of PcGTs/TSGs, leading to increased mammary cancer in F1-F4 generation offspring, will be investigated by treating F1-F4 generation mice with histone deacetylase (HDAC) and DNMT inhibitors. Our preliminary study indicates that an exposure to HDAC+DNMT inhibitors during adult life completely reverses the increase in mammary cancer risk in in utero estrogen exposed mice, but whether these exposures reverse increased DNA methylation and increased mammary tumorigenesis in F2-F4 generations of estrogen exposed dams, is not known. Finally, as there is currently no way of knowing who might have been exposed to high in utero estrogenic environment, we will study whether these individuals can be identified by determining E2/ER regulated miRNA profile in the peripheral blood.